OBJECTIVETo clone and characterize the DNA and cDNA sequences of phenylalanine ammonia-lyase gene (PAL) from Fagopyrum dibotrys, and investigate the biological activity of the obtained PAL.

METHODUsing homology cloning and RT-PCR techniques, the DNA and full-length cDNA sequences of PAL gene were amplified from F. dibotrys. The obtained sequences were analyzed by bioinformatics software. The ORF of PAL gene was cloned into expression vector pET-30b(+) and transformed into Escherichia coli BL21 (DE3) for expression the recombined protein. The catalytic activity of the recombined protein was determined by Spectrophotometer and thin layer chromatography (TLC) methods.

RESULTThe DNA sequence of PAL gene (designated as FdPAL, GenBank accession number: HM628904) was 2 583 bp in size, of which consisted two extrons and a single intron, and the full-length cDNA of FdPAL was 2 169 bp in size, which contained an ORF. The deduced protein of FdPAL contained 722 amino acids with calculated molecular weight (MW) of 78.31 kDa and an isoelectric point (pI) of 5.94. The SDS-PAGE results showed that the molecular weight of recombinant FdPAL protein was 75.37 kDa, which is consistent with the predictions. After 4 hours of induction, the enzymatic specific activity of FdPAL reached the summit, up to 4 386 nmol x g(-1) x min(-1). The reaction products were also identified by TLC, using L-Phe and trans-cinnamic acid as the internal standard.

CONCLUSIONThe PAL gene (both DNA sequence and full-length cDNA sequence) was cloned from F. dibotrys, and it has the same classic characters as other PALs in plants. The recombinant FdPAL was efficiently expressed in E. coli and had the activity for catalyzing the conversion from L-phenylalanine to cinnamic acid.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fagopyrum ; classification ; genetics ; Molecular Sequence Data ; Phenylalanine Ammonia-Lyase ; chemistry ; genetics ; metabolism ; Phylogeny ; Protein Conformation ; Recombinant Proteins ; chemistry ; genetics ; metabolism

Objective To analyze fungal isolates from patients with superficial fungal infections during 1960-2006.Methods Fungal strains isolated from patients with superficial (mucocutaneous and cutaneous)fungal infections and identified in the Medical Mycology Clinical Laboratory,Department of Dermatology and Venereology,Union Hospital,from 1960 to 2006 (data from September 1991 to July 1992 were unavailable),were subjected to a classification and statistical analysis.Clinical samples for mycological examination were taken from outpatients or inpatients of different departments in hospitals of Hubei province and surrounding areas.Morphological,physiological and biochemical methods were applied for species identification.Results A total of 11 989 Candida strains were isolated,which belonged to 23 species and 16 genera.They fell into 3 groups,i.e.,dermatophytes,Candida and yeasts (including Malassezia),and non-dermatophyte moulds.Since 287 strains of moulds were suspected to be contaminating fungi,11 702 residual isolates were analyzed.Of the analyzed isolates,Candida species (5642/11 702,48.2％ )and dermatophytes (5279/11 702,45.1％ )predominated,followed by yeasts (449/11 702,3.8％) and Malassezia species (332/11 702,2.8％).The most frequently isolated species was Trichophyton rubrum (3865/11 702,33.0％),Candida albicans (3110/11 702,26.6％ ) and non-albicans Candida species (2532/11 702,21.6％ ).Dermatophyte strains were mostly isolated from lesions of smooth skin with an exception of palmoplantar and interdigit regions (1787/5279,37.7％).The most common dermatophyte species was Trichophyton rubrum,followed by Trichophyton violanceum.Candida was mainly isolated from mucous membrane lesions (4099/5642,72.7％),with Candida albicans being the predominant species.Conclusions Candida species and dermatophytes predominate in patients with superficial fungal infections during 1960-2006,with Trichophyton rubrum being the most common species.